Penetration of analogues of H2O and CO2 in proteins studied by room temperature phosphorescence of tryptophan.

The influence of the protein matrix on the reactivity of external molecules with a species buried within the protein interior is considered in two general ways: (1) there may be structural fluctuations that allow for the diffusive penetration of the small molecules and/or (2) the external molecule may react over a distance. As a means to study the protein matrix, a reactive species within the protein can be formed by exciting tryptophan to the triplet state, and then the reaction of the triplet-state molecule with an external molecule can be monitored by a decrease in phosphorescence. In this work, the quenching ability (i.e., reactivity) was examined for H2S, CS2, and NO2- acting on tryptophan phosphorescence in parvalbumin, azurin, horse liver alcohol dehydrogenase, and alkaline phosphatase. A comparison of charged versus uncharged quenchers (H2S vs SH- and CS2 vs NO2-) reveals that the uncharged molecules are much more effective than charged species in quenching the phosphorescence of fully buried tryptophan, whereas the quenching for exposed tryptophan is relatively independent of the charge of the quencher. This is consistent with the view that uncharged triatomic molecules can penetrate the protein matrix to some extent. The energies of activation of the quenching reaction are low for the charged quenchers and higher for the uncharged CS2. A model is presented in which the quenchability of a buried tryptophan is inversely related to the distance from the surface when diffusion through the protein is the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of synaptosomal enzymes by local anesthetics

The effects of tertiary amine local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and chlorpromazine were investigated for three enzyme activities associated with rat brain synaptosomal membranes, i.e., (Na⁺ + K⁺)-ATPase (ouabain-sensitive), Mg²⁺-ATPase (ouabain-insensitive) and acetylcholinesterase. Approximately the same concentrations of each agent gave 50% inhibition of both ATPase, for example 7.9 and 10 mM tetracaine for Mg²⁺-ATPase and (Na⁺ + K⁺)-ATPase, respectively; these concentrations are 10-fold higher than required for inhibition of mitochondrial F₁-ATPase. The relative inhibitory potency of the several agents was proportional to their octanol/water partition coefficients. Acetylcholinesterase was inhibited by all agents tested, but the ester anesthetics (procaine and tetracaine) were considerably more potent than the others after correction for partition coefficient differences. For tetracaine, 0.18 mM gave 50% inhibition and showed competitive inhibition on a Lineweaver-Burk plot, but for dibucaine a mixed type of inhibition was observed, and 0.63 mM was required for 50% inhibition. Tetracaine evidently binds at the active site, and dibucaine at the peripheral or modulator site, on this enzyme.     

Tryptophanyl-tRNA synthetase is a major soluble protein species in bovine pancreas

Besides their central role in protein synthesis, aminoacyl-tRNA synthetases have been found or thought to be involved in other processes. We present here a study showing that tryptophanyl-tRNA synthetase has a surprising tissular distribution. Indeed, immunochemical determinations showed that in several bovine organs such as liver, kidney and heart, tryptophanyl-tRNA synthetase constitutes, as expected, about 0.02% of soluble proteins. In spleen, brain cortex, stomach, cerebellum or duodenum, this amount is about 10-times higher, and in pancreas it is 100-fold. There is no correlation between these amounts and the RNA content of the organs. Moreover, the concentration of another aminoacyl-tRNA synthetase (methionyl-tRNA synthetase) is higher in liver than in pancreas, while the amount of tRNATrp is not higher in pancreas than in liver as compared to other tRNAs. Among several interpretations, it is possible that tryptophanyl-tRNA synthetase is involved in a function other than tRNA aminoacylation. This unknown function would be specific to the differentiated organs, since fetal cerebellum and fetal pancreas contain the same amount of tryptophanyl-tRNA synthetase as adult liver.     

Symbiosis research, technology, and education: Proceedings of the 6th International Symbiosis Society Congress held in Madison Wisconsin, USA, August 2009

Symbiosis, the intimate association between two or more organisms, is a fundamental component of biological systems. Our ability to understand the processes involved in the establishment and function of Symbiosis has critical consequences for the health of humans and the world we live in. For example, a deeper understanding of how legumes and insects have harnessed the nitrogen-fixing capacity of microbes can pave the way toward novel strategies to decrease fertilizer use. Also, using insect models to elucidate links between diet, gut microbiota, and toxin sensitivity not only has implications for biological control strategies, but also will lend insights into similar links in the human gut ecosystem. These types of ideas were presented and discussed at the 6th International Symbiosis Society Congress held in Madison, Wisconsin August, 2009. Over 300 participants from 20 countries attended the 7-day event, which featured cutting-edge symbiosis research from many different perspectives and disciplines. The conference was organized thematically, with oral sessions focused on Evolution, Ecology, Metabolism, the Host-Microbe Interface, Threats to Earth Systems, Symbiosis Models and the Human Microbiome, Viruses and Organelles, and Symbiosis Education. World-renowned scientists, post-doctoral fellows, and students were given the opportunity to describe their most recent discoveries. Session chairs provided overviews of their programs which highlight how the comparative analysis of different systems reveal common trends underlying symbiotic associations, what tools and theory are being developed that may be applied more broadly in symbiosis research, how symbiosis research contributing solutions to global issues such as emerging antibiotic resistance, a need for alternative energy sources, the pursuit of sustainable agriculture and natural resources, and how symbiotic systems are ideal for educating people about the fascinating natural world around us. The following paragraphs provide an overview of the research and discussions that took place during the congress.     

Evidence for a Ca2+-independent association between calpain II and phospholipid vesicles

Possible interactions between calpain II and phospholipids such as phosphatidylinositol, phosphatidylserine and phosphatidylcholine were studied using fluorescence and gel filtration techniques. Changes in fluorescence intensity of purified calpain II show that the enzyme strongly interacts with phosphatidylinositol and phosphatidylserine and to a lesser extent with phosphatidylcholine. These results are corroborated by the gel filtration technique which permits the isolation of the enzyme phospholipid complex. Association between calpain II and various phospholipid vesicles can occur in the absence of calcium. Such binding occurs without any observable change of the molecular mass of the two subunits on SDS-polyacrylamide gel electrophoresis.     

Thematic Review Series: New Lipid and Lipoprotein Targets for the Treatment of Cardiometabolic Diseases: Niacin,an old drug with a new twist

Niacin (nicotinic acid) has been used for decades as a lipid-lowering drug. The clinical use of niacin to treat dyslipidemic conditions is limited by its side effects. Niacin, along with fibrates, are the only approved drugs which elevate high density lipoprotein cholesterol (HDLc) along with its effects on low density lipoprotein cholesterol (LDLc) and triglycerides. Whether niacin has a beneficial role in lowering cardiovascular risk on the background of well-controlled LDLc has not been established. In fact, it remains unclear whether niacin, either in the setting of well-controlled LDLc or in combination with other lipid-lowering agents, confers any therapeutic benefit and if so, by which mechanism. The results of recent trials reject the hypothesis that simply raising HDLc is cardioprotective. However, in the case of the clinical trials, structural limitations of trial design complicate their interpretation. This is also true of the most recent Heart Protection Study 2-Treatment of HDLc to Reduce the Incidence of Vascular Events (HPS2-THRIVE) trial in which niacin is combined with an antagonist of the D prostanoid (DP) receptor. Human genetic studies have also questioned the relationship between cardiovascular benefit and HDLc. It remains to be determined whether niacin may have clinical utility in particular subgroups, such as statin intolerant patients with hypercholesterolemia or those who cannot achieve a sufficient reduction in LDLc. It also is unclear whether a potentially beneficial effect of niacin is confounded by DP antagonism in HPS2-THRIVE.     

Comparison of 26 Sphingomonad Genomes Reveals Diverse Environmental Adaptations and Biodegradative Capabilities

Sphingomonads comprise a physiologically versatile group within the Alphaproteobacteria that includes strains of interest for biotechnology, human health, and environmental nutrient cycling. In this study, we compared 26 sphingomonad genome sequences to gain insight into their ecology, metabolic versatility, and environmental adaptations. Our multilocus phylogenetic and average amino acid identity (AAI) analyses confirm that Sphingomonas, Sphingobium, Sphingopyxis, and Novosphingobium are well-resolved monophyletic groups with the exception of Sphingomonas sp. strain SKA58, which we propose belongs to the genus Sphingobium. Our pan-genomic analysis of sphingomonads reveals numerous species-specific open reading frames (ORFs) but few signatures of genus-specific cores. The organization and coding potential of the sphingomonad genomes appear to be highly variable, and plasmid-mediated gene transfer and chromosome-plasmid recombination, together with prophage- and transposon-mediated rearrangements, appear to play prominent roles in the genome evolution of this group. We find that many of the sphingomonad genomes encode numerous oxygenases and glycoside hydrolases, which are likely responsible for their ability to degrade various recalcitrant aromatic compounds and polysaccharides, respectively. Many of these enzymes are encoded on megaplasmids, suggesting that they may be readily transferred between species. We also identified enzymes putatively used for the catabolism of sulfonate and nitroaromatic compounds in many of the genomes, suggesting that plant-based compounds or chemical contaminants may be sources of nitrogen and sulfur. Many of these sphingomonads appear to be adapted to oligotrophic environments, but several contain genomic features indicative of host associations. Our work provides a basis for understanding the ecological strategies employed by sphingomonads and their role in environmental nutrient cycling.     

Blood Microbiota Dysbiosis Is Associated with the Onset of Cardiovascular Events in a Large General Population: The D.E.S.I.R. Study

Aim

We recently described a human blood microbiome and a connection between this microbiome and the onset of diabetes. The aim of the current study was to assess the association between blood microbiota and incident cardiovascular disease.

Methods and Results

D.E.S.I.R. is a longitudinal study with the primary aim of describing the natural history of the metabolic syndrome and its complications. Participants were evaluated at inclusion and at 3-, 6-, and 9-yearly follow-up visits. The 16S ribosomal DNA bacterial gene sequence, that is common to the vast majority of bacteria (Eubac) and a sequence that mostly represents Proteobacteria (Pbac), were measured in blood collected at baseline from 3936 participants. 73 incident cases of acute cardiovascular events, including 30 myocardial infarctions were recorded. Eubac was positively correlated with Pbac (r = 0.59; P<0.0001). In those destined to have cardiovascular complications, Eubac was lower (0.14±0.26 vs 0.12±0.29 ng/µl; P = 0.02) whereas a non significant increase in Pbac was observed. In multivariate Cox analysis, Eubac was inversely correlated with the onset of cardiovascular complications, (hazards ratio 0.50 95% CI 0.35–0.70) whereas Pbac (1.56, 95%CI 1.12–2.15) was directly correlated.

Conclusion

Pbac and Eubac were shown to be independent markers of the risk of cardiovascular disease. This finding is evidence for the new concept of the role played by blood microbiota dysbiosis on atherothrombotic disease. This concept may help to elucidate the relation between bacteria and cardiovascular disease.     

The Genome Sequences of Cellulomonas fimi and “Cellvibrio gilvus” Reveal the Cellulolytic Strategies of Two Facultative Anaerobes,Transfer of “Cellvibrio gilvus” to the Genus Cellulomonas,and Proposal of Cellulomonas gilvus sp. nov

Actinobacteria in the genus Cellulomonas are the only known and reported cellulolytic facultative anaerobes. To better understand the cellulolytic strategy employed by these bacteria, we sequenced the genome of the Cellulomonas fimi ATCC 484^T. For comparative purposes, we also sequenced the genome of the aerobic cellulolytic “Cellvibrio gilvus” ATCC 13127^T. An initial analysis of these genomes using phylogenetic and whole-genome comparison revealed that “Cellvibrio gilvus” belongs to the genus Cellulomonas. We thus propose to assign “Cellvibrio gilvus” to the genus Cellulomonas. A comparative genomics analysis between these two Cellulomonas genome sequences and the recently completed genome for Cellulomonas flavigena ATCC 482^T showed that these cellulomonads do not encode cellulosomes but appear to degrade cellulose by secreting multi-domain glycoside hydrolases. Despite the minimal number of carbohydrate-active enzymes encoded by these genomes, as compared to other known cellulolytic organisms, these bacteria were found to be proficient at degrading and utilizing a diverse set of carbohydrates, including crystalline cellulose. Moreover, they also encode for proteins required for the fermentation of hexose and xylose sugars into products such as ethanol. Finally, we found relatively few significant differences between the predicted carbohydrate-active enzymes encoded by these Cellulomonas genomes, in contrast to previous studies reporting differences in physiological approaches for carbohydrate degradation. Our sequencing and analysis of these genomes sheds light onto the mechanism through which these facultative anaerobes degrade cellulose, suggesting that the sequenced cellulomonads use secreted, multidomain enzymes to degrade cellulose in a way that is distinct from known anaerobic cellulolytic strategies.